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Protocols for the High - Barcode of Life

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					  PROTOCOLS FOR THE
HIGH-VOLUME ASSEMBLY
   OF DNA BARCODES


   Mehrdad Hajibabaei
   Barcode of Life Initiative
    University of Guelph
          Canada
                                     Barcoding Animals


Gaining Barcode Closure for Animals



               10 million species
                       x
               10 barcodes each
                       =
              100 million barcodes
                                     Barcoding Animals


Barcodes in Perspective



             100 million barcodes
                      =
             65 billion base pairs
                                                                                    Barcoding Animals


The Time Frame

                                10000
     Time to Complete (Years)


                                1000
                                                              200 x 50K sequences per year

                                 100
                                                                                  10 years to
                                                                                  completion

                                  10


                                   1
                                        0.01 M




                                                 0.1 M




                                                               1M




                                                                           10 M




                                                                                       100 M
                                                         Sequences/Year
                                     Barcoding Animals


Logistical Challenges


   How do we increase production rates,
    lower costs and speed analysis?



   How do we recover barcodes from
    archival specimens?
                                               The Barcoding Process


The Analytical Chain




  Specimen or Tissue Sample


                                         DNA Barcode




         Extract DNA          PCR COI   Sequence COI
                                      The Barcoding Process


Model 1: Core Facility

                   Specimen




                  Core Facility
             50-100K Barcodes/Year
 Specimen             $1M                         Specimen




                                     * Core Facility receives
                                       specimen samples
                   Specimen
                                              The Barcoding Process


Model 2: Satellite Labs


                        Satellite Lab $20K




                     Sequencing Facility
                          $800K
Satellite Lab $20K                                Satellite Lab $20K




                        Satellite Lab $20K   * Sequencing Facility
                                               receives PCR product
                                  The Barcoding Process


The Guelph Analytical Node

                           Sourcing Specimens

  Guelph Analytical Node   Extracting DNA

                           DNA Amplification

                           Screening PCR Products

                           Sequencing

         Barcode
          of Life
         Database
                                                                       Sourcing Specimens


Sourcing Specimens

                                              moderately
                                              abundant


                               rare                              very
  number of species




                      20                                       abundant

                      15


                      10


                      5



                           1     2    4   8   16 32   64 128 256 512
                                          log abundance
                              Sourcing Specimens


Specimen Submission Package
                 DNA Extraction


DNA Extraction
                                          DNA Extraction


Sample Material
 Destructive tissue
   sampling is the rule…   But the amount is small
                                               DNA Extraction


Non-Destructive Methods
Non-destructive methods are available




           Minute specimens             Type specimens
                                DNA Extraction


Two Favored Methods


    Method      Cost ($)   Sensitivity

    Chelex       0.10          +

   Membrane      1.50         +++
                                 DNA Amplification


DNA Amplification
 Primer Design      Optimized Reaction
                        DNA Amplification


Primer Design

                  100


                  75
    PCR Success




                  50


                  25


                   0
                            DNA Amplification


Optimized Reaction


 Removes the need for PCR
  cleanup

 Lower cost by decreasing
   reaction volume
                         Screening PCR Products


Screening PCR Products
                      Screening PCR Products


A Fast-Gel Approach
                           Screening PCR Products


A Micro-Fluidic Approach
                                       Screening PCR Products


A Comparison of Methods



                 Standard   Fast-Gel     Micro-fluidic
                 Agarose
Capital            0.5K      0.5K          100-250K

Maintence/Year     N/A        N/A           15-30K

Cost/Sample        0.03       0.3           0.5- 0.8

Time/96(min)       45         15               60
                                            Sequencing


Sequencing
 Sequencing Reaction   Sequence Alignment
 and Clean up
                                   Sequencing


Sequencing Reaction and Clean up

     1000
   standard
   reactions




   $10,584
                                 Sequencing


Sequence Alignment

 Sequencher®         SeqScape®
                      Data Management


The Need for a LIMS



                              NCBI




                              BoLD
                                        Cost Analysis


Cost Analysis of Barcoding

  Analysis                   Cost ($)
  DNA Extraction              0.34
  PCR                         0.24
  Fast-Gel                    0.35
  Sequencing X2               1.04
  Cleanup X2                  0.32
  Sequence Run X2             0.40
  Operational Costs           2.50
  Total                       5.19
                                    Archived Specimens


Barcodes from Archived Specimens




                                Time




                        Barcode fragmentation
                                                      Archived Specimens


Full Barcodes versus Mini-Barcodes

                        Full Barcode   Mini-Barcode
        100


              75
  % Success




              50


              25


              0
                   3-15 years              15-30 years
                    N=56                      N=41
                                 Archived Specimens


Immortalizing and Rescuing DNA


 Whole genome
 amplification


 DNA repair
                      h
                                                              Archived Specimens


Whole Genome Amplification
    1000-fold amplification of whole genome

Genomic DNA            Amplified Genomic DNA              Amplified Barcode
                                                               Region




       Whole Genome                            Standard
       Amplification                             PCR
                                                          Archived Specimens


DNA Repair
 130 DNA repair genes in the human genome
 DNA repair enzymes
 Augmented PCR cocktails: i.e. Restorase®

   Fragmented DNA            Repaired DNA         Amplified Barcode
       Barcode                                         Region




                DNA repair                  Standard
                 protocol                     PCR
                                                                                 Archived Specimens


Early Restorase I Results
                                                                                       2002
                                        Restorase I         Regular Taq

                 100
 % PCR Success




                 50




                  0
                             )




                                             )




                                                             )




                                                                             )
                           bp




                                           bp




                                                           bp




                                                                           bp
                        00




                                        00




                                                        00




                                                                        50             1930
                       (7




                                       (6




                                                       (4




                                                                       (3
                   1




                                   2




                                                   3




                                                                   4
               R




                                   R




                                                   R




                                                                   R
             PC




                                 PC




                                                 PC




                                                                 PC



                                                                                  Range of specimen
                                                                                   collection dates
                                                                                     Archived Specimens


Early Restorase II Results
                                                                                           2002
                             Restorase II        Diamond Taq           Regular Taq
                 100
 % PCR Success




                  50




                   0
                             )




                                             )




                                                             )




                                                                             )
                           bp




                                           bp




                                                           bp




                                                                           bp
                        00




                                        00




                                                        00




                                                                        50
                                                                                           1930
                       (7




                                       (6




                                                       (4




                                                                       (3
                   1




                                   2




                                                   3




                                                                   4
                   R




                                   R




                                                   R




                                                                   R
                 PC




                                 PC




                                                 PC




                                                                 PC


                                                                                      Range of specimen
                                                                                       collection dates
                                       Partnerships


Barcodes for Biodiversity Assessment
                                     On the Horizon


On the Horizon
           The Analytical Keychain
                                      On the Horizon


On the Near Horizon
 Speed – Specimen to
                            ID Time
 barcode-based ID in
 3 hours


 Volume – 500K+ sequences
 per-year via automation
                                                    Acknowledgments


Acknowledgements

     Laboratory                            Database
   Jeremy DeWaard                      Sujeevan Ratnasingham
    Nataly Ivanova                           Rob Dooh
    Stephanie Kirk
     Janet Topan
    Angela Hollis        Paul Hebert        Funding &
                                             Support
                                       Gordon and Betty Moore
     Collections                            Foundation
      Dan Janzen                              NSERC
     John Burns
                                       Canada Research Chairs
  Jean-Francois Landry                        Program

				
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posted:8/23/2011
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